CD147 Blockade As a Potential and Novel Treatment of Graft Rejection

MOLECULAR MEDICINE REPORTS 16: 4593-4602, 2017

CD147 blockade as a potential and novel treatment of graft rejection

JING LUAN1,2*, YU ZHAO1,2*, YANG ZHANG1,2, JINLIN MIAO1,3, JIA LI1,3, ZHI‑NAN CHEN1,2 and PING ZHU1,3

1National Translational Science Center for Molecular Medicine, The Fourth Military Medical University; 2Cell Engineering Research Center and Department of Cell Biology, The Fourth Military Medical University; 3Department of Clinical Immunology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China

Received March 16, 2017; Accepted August 1, 2017

DOI: 10.3892/mmr.2017.7201

Abstract. Cluster of differentiation (CD)147 is highly involved Furthermore, the level of inflammatory cell infiltration in in the T cell activation process. High CD147 expression is transplanted skin was reduced. CD147 blockade decreased observed on the surfaces of activated T cells, particularly the serum levels of interleukin (IL)‑17 and the proportions of CD4+ T cells. In organ transplantation, it is important to peripheral blood CD4+ and CD8+ memory T cells. The data prevent graft rejection resulting from the excessive activa- demonstrated that CD147 blockade suppressed skin graft tion of T cells, particularly CD4+ T cells, which exhibit a rejection, primarily by suppressing CD4+T and memory key role in amplifying the immune response. The present T cell proliferation, indicating that CD147 exhibits great study aimed to investigate the effects of CD147 blockade potential as a target of immunosuppressant drugs. in vitro and in vivo and used a transplant rejection system to assess the feasibility of utilizing CD147 antibody‑based Introduction immunosuppressant drugs for the treatment of graft rejection. The effects of CD147 antibodies were evaluated on Transplantation remains the best treatment option for lymphocyte proliferation stimulated by phytohemagglutinin correcting certain types of organ failure and tissue damage, or CD3/CD28 magnetic beads and in a one‑way mixed e.g., that of the kidneys, heart, and lungs (1). In organ transplan- lymphocyte reaction (MLR) system in vitro. For the in vivo tation, it is always a main goal to prevent rejection. However, analysis, an allogeneic skin transplantation mouse model was the long‑term use of globally nonspecific immunosuppressant used. CD147 antibodies were effective against lymphocytes, drugs to prevent rejection is associated with serious risks, particularly CD4+T lymphocytes, and were additionally including a weakened immune system, infection, and cancer, effective in the one‑way MLR system. In the allogeneic skin as well as graft loss due to their toxicity (1,2). Thus, identifying transplantation mouse model, the survival of transplanted safe, target‑specific immunosuppressive agents for promoting skin was extended in the CD147 antibody‑treated group. allograft survival is a high priority. Acute rejection is clinically the most common and is mainly mediated by cellular immune responses; this type of rejection can occur any time from a week to months after Correspondence to: Dr Zhi‑Nan Chen or Professor Ping Zhu, transplantation. T cells, especially CD4+ T cells from the National Translational Science Center for Molecular Medicine, The recipient, play a key role in acute transplantation rejection. Fourth Military Medical University, 169 Changle West Road, Xi'an, As a trigger, CD4+ T cells amplify the downstream signal. Shaanxi 710032, P.R. China By recognizing HLA‑Ⅱ antigens and interacting with them, E‑mail: [email protected] CD4+ T cells cause antigen‑presenting cells to release inter- E‑mail: [email protected] leukin (IL)‑1, which could promote CD4+ T cells releasing IL‑2. IL‑2 can, in turn, promote CD4+ T cell proliferation * Contributed equally and provide an auxiliary signal for CTL cell differentiation. Furthermore, CD4+ T cells produce IL‑4 and IL‑5 to promote Abbreviations: CCK‑8, Cell Counting kit 8; FITC, fluorescein B cell differentiation and the secretion of antibodies against isothiocyanate; FK506, tacrolimus; IFN‑γ, interferon γ; IL‑17, the graft to participate in the rejection reaction. Different interleukin 17; IL‑4, interleukin 4; mAb, monoclonal antibody; MHC, major histocompatibility complex; MLR, mixed lymphocyte CD4+ T cell subsets execute different functions during reaction; MS, multiple sclerosis; PBMCs, peripheral blood transplant rejection. There have been studies supporting mononuclear cells; PE, phycoerythrin; PI, propidium iodide; RA, the notion that the balance of the Th1/Th2 cell proportion rheumatoid arthritis is a determining factor in this process. Th1 cells mainly participate in graft rejection, especially in acute rejection; Key words: CD147 antibody, immunosuppressant, graft rejection, in contrast, Th2 cell activation leads to the formation of treatment transplantation tolerance (3,4). Furthermore, cytokines such as Th1‑related interferon (IFN)‑γ and Th2‑related IL‑2, IL‑4 4594 LUAN et al: CD147 BLOCKADE SHOWS GREAT POTENTIAL FOR THE TREATMENT OF GRAFT REJECTION and IL‑10 participate in the development of rejection (5). As not meet the criteria for FDA approval; however, it did research continues, CD4+ T cell subsets, e.g., Th17 and Treg, show activity against CD147, which suggests that CD147 is are considered to be the two main types involved in allograft an effective target for the treatment of GVHD (25‑27). The rejection and survival (6,7). effect of CD147 blockade on allograft rejection has never In addition to the above‑mentioned mechanisms of CD4+ been investigated before. Thus, this study was performed to T cell subsets, memory T cells (both CD4+ and CD8+) have investigate whether the blockade of CD147 can inhibit the been found to be involved in development of the rejection rejection reaction and to determine whether CD147 antibodies reaction (8‑10). In a primate model, the elimination of could be developed as specific immunosuppressors for the memory T cells has been shown to induce the body's immune graft rejection response. tolerance (11). It has also been reported that infusion of sensitized memory CD4+ T cells produces acute rejection Materials and methods in mice that have undergone abdominal heterotopic heart transplantation. In addition, infusion of sensitized memory Antibodies and reagents. Humanized mAbs (5A12) against CD4+ T cells has been shown to significantly shorten the CD147 were generated and identified in our laboratory. survival times of transplanted hearts, and this effect cannot A purified human IgG1 isotype control antibody (Clone: be reversed by commonly used immunosuppressants (12,13). ET901) was purchased from BioLegend, Inc. (San Diego, Clinical studies have shown that the prognoses of patients CA, USA). An anti‑mouse CD147 functional‑grade purified who have undergone liver transplantation are dependent on antibody (Clone: RL73) and its isotype control (Clone: eBR2a) patient CD8+ T memory cell status (14). The results of an were purchased from eBioscience (San Diego, CA, USA). in vivo study have shown that endogenous donor‑reactive Tacrolimus (FK506) was purchased from KeHao (Wuhan, CD8+ memory T cells infiltrate the transplanted hearts Hubei, China). Fluorescein isothiocyanate (FITC)‑conjugated of mice within a few h after reperfusion and secrete mouse anti‑human CD4 and phycoerythrin (PE)‑conjugated IFN‑γ, causing inflammation (15). It is well known that mouse anti‑human CD3 antibodies, Foxp3 staining buffer antibody‑mediated T cell depletion is one of the most potent and a cell activation cocktail used for intracellular staining immunosuppressant therapies. This therapy is increasingly were purchased from BioLegend. PE‑Cy7‑conjugated rat used as an induction therapy in organ transplantation (16). anti‑mouse IFN‑γ, PE‑conjugated rat anti‑mouse IL‑4, However, T cell homeostasis after depletion therapy leads to Alexa 647‑conjugated rat anti‑mouse IL‑17, FITC‑conjugated a predominance of memory T cells, which are more potent rat anti‑mouse CD4, allophycocynanin‑H7‑conjugated rat than naïve T cells in mediating graft rejection and present a anti‑mouse CD8, PE‑Cy7‑conjugated rat anti‑mouse CD44, major obstacle to achieving tolerance (17,18). BUV737‑conjugated rat anti‑mouse CD62 L, allophyco CD147 is a cell‑surface glycosylated transmembrane cyanin‑conjugated rat anti‑mouse CD25, and PE‑conjugated protein that belongs to the immunoglobulin superfamily. This rat anti‑mouse Foxp3 antibodies were purchased from BD protein serves multiple biological functions and is widely Biosciences (San Diego, CA, USA). expressed in many tissues and cell types, such as normal brain tissue, tracheal, lung, and breast tissues, as well as Cell isolation. Human peripheral blood was obtained from lymphocytes and neutrophils (19,20). High CD147 expression healthy volunteers who provided informed consent. In brief, is involved in many different diseases. In the immune system, lymphocytes were isolated by density gradient centrifugation CD147 participates in different stages of T cell activity, over Lymphocyte Separation Medium (MP Biomedicals, including development, activation, proliferation, migration, LLC, Santa Ana, CA, USA) according to the manufacturer's and adhesion (19,21,22). It is worth mentioning that CD147 instructions. CD4+ T cells were isolated by negative selec- has been identified as a T cell activation‑related antigen (M6) tion using a magnetic cell separation system according to expressed in phytohemagglutinin (PHA)‑activated T lympho- the manufacturer's instructions (Miltenyi Biotec, Bergisch cytes (23). In a previous study at our institute, we found that Gladbach, Germany). The isolated cells were then washed CD147 participated in immunological synapse formation by once and resuspended in phosphate‑buffered saline (PBS). co‑localizing with CD48 molecules on the surface of T cells. After incubation with PE‑conjugated mouse anti‑human CD3 In addition, blocking CD147 decreased intracellular calcium and FITC‑conjugated mouse anti‑human CD4 antibodies, the mobilization and affected protein tyrosine phosphorylation purities were determined by flow cytometry (FACSCalibur; upon CD3/TCR stimulation, all of which are very important BD Biosciences). The purity of CD3+ CD4+ T cells consis- in the process of T cell activation. Interestingly, CD147 tently ranged from 92‑95%. expression was clearly increased upon T cell activation, and this trend was the most obvious in CD4+ T cell subsets (24). Cell proliferation, apoptosis, and cytokine production. This information suggests that CD147 blockade is a poten- Human lymphocytes and CD4+ T cells were separately tial way to inhibit the function of T cells, especially CD4+ plated in 96‑well plates at a density of 3x105 cells/well in T cells. 200 µl of RPMI 1640 supplemented with 10% fetal bovine ABX‑CBL, also known as gavilimomab, is a hybri serum. Then, the cells were stimulated with PHA (2 µg/ml; doma‑generated murine IgM monoclonal antibody (mAb) Sigma, St. Louis, MO, USA) or co‑cultured with CD3/CD28 against CD147. Studies using ABX‑CBL for the treatment magnetic beads (at a bead‑to‑cell ratio of 1:1; Gibco Life of steroid‑resistant acute GVHD have shown encouraging Technologies, Carlsbad, CA, USA) with or without the addi- results. ABX‑CBL did not yield improvements in outcomes tion of 5A12 (six wells were performed in parallel in each compared with anti‑thymocyte globulin and therefore did group). Lymphocyte proliferation was measured using Cell MOLECULAR MEDICINE REPORTS 16: 4593-4602, 2017 4595

Counting kit 8 (CCK‑8) assay (Engreen Biosystem, Beijing, Blood was then collected from the orbital sinus. Plasma was China) according to the manufacturer's instructions. Cell obtained by centrifugation and stored at ‑20˚C for later use. apoptosis was evaluated by flow cytometry (FACSCalibur; Cytokine production was assessed using a MILLIPLEX BD Biosciences) using an FITC Annexin V Apoptosis MAP kit (EMD Millipore Corporation, Billerica, MA, USA) Detection kit with propidium iodide (PI; BioLegend, Inc.). according to the manufacturer's instructions. Splenocytes In brief, cells were washed twice and then resuspended at and peripheral blood were stained with antibodies according a concentration of 0.5‑1.0x107 cells/ml. Next, 100 µl of cell to conventional treatment methods and then immediately suspension was transferred to each well and incubated with assessed using an LSRFortessa X‑20 flow cytometer (BD 5 µl of FITC Annexin V and 10 µl of PI at room tempera- Biosciences). Skin grafts were harvested and fixed in 4% ture for 15 min. The samples were then ready for testing. formalin for histological studies. Histological analysis of Cells were collected by flow cytometry (FACSCalibur; the skin grafts was performed by staining 5‑mm sections BD Biosciences). of paraffin‑embedded tissues with hematoxylin and eosin. Inflammatory cells were counted by examining 10 different One‑way mixed lymphocyte reaction (MLR). The experimental visual fields under a microscope at magnification, x400 in a method used here has been previously described (28,29). blinded manner. Peripheral blood mononuclear cells (PBMCs), i.e., lymphocytes, from human volunteers were used as responder cells, Statistical analysis. The results presented are representative and splenocytes from C57BL/6 mice were used as stimulator of a minimum of three experiments and are expressed as the cells. The lymphocytes were suspended in RPMI 1640 mean ± standard deviation unless otherwise stated. Multiple supplemented with 10% fetal bovine serum at a density of comparisons were performed with one‑way ANOVA. Graft 1.5x106 cells/ml and marked as sample A. Two‑thirds of survivals were analyzed by the Kaplan‑Meier method using the splenocytes were removed and incubated with mito- the log‑rank test. For analysis of T cell responses with flow mycin C (Sigma) at a final concentration of 25 µg/ml in cytometry method, nonparametric Mann‑Whitney U tests RPMI 1640 (without fetal bovine serum) at 37˚C for 30 min. were performed. GraphPad software (GraphPad Software, The remaining splenocytes were used as a control. After Inc. La Jolla, CA, USA) was used for the above analyses, and incubation, the splenocytes were washed twice with PBS, P<0.05 was considered significant. resuspended at the same density of 1.5x106 cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum and marked Results as sample B. Samples A and B were suspended and mixed at a ratio of 1:1; then, 200 µl of the cell suspension was pipetted Proliferation of CD4+ T cells from human peripheral blood into round‑bottomed 96‑well plates. Cells were cultured with was inhibited by 5A12. We have previously reported that or without 5A12 for 96 h at 37˚C and 5% CO2 in a humidified CD147 expression is clearly increased upon T cell activation, atmosphere. Then, lymphocyte proliferation and cytokine especially in CD4+ T cell subsets (24). We first verified the production were detected using human IFN‑γ, IL‑4 and IL‑17 effects of the humanized CD147 mAb 5A12 on the prolife ELISA kits (Dakewe Bio Engineering Co., Ltd., Shenzhen, ration of CD4+ T cells isolated from the peripheral blood of China). healthy people. When CD4+ T cells were stimulated with PHA or CD3/CD28 magnetic beads, marked proliferation Animals. Male C57BL/6 (H‑2b) and BALB/c (H‑2d) mice was induced. The addition of 5A12 to the cultures strongly were obtained from Beijing Vital River Laboratory Animal inhibited this proliferation by 28 and 77% in the PHA‑ and Technology Co., Ltd. (Beijing, China) and housed in a CD3/CD28‑stimulated groups, while the resting lymphocytes specific pathogen‑free facility in microisolator cages at The were unaffected (Fig. 1A and B). In addition, 5A12 inhibited Fourth Military Medical University laboratory animal center. proliferation in a dose‑dependent manner; the minimum All animals used were 8‑10 weeks old. All protocols were significant concentrations for PHA‑stimulated and CD3/CD28 approved by the institutional Ethics Committee on animal magnetic bead‑stimulated CD4+ T cells were 100 µg/ml and use of The Fourth Military Medical University of Science 50 µg/ml, respectively (data not shown). Compared with PHA, and Technology. The skin allografts were placed using a stan- CD3/CD28 stimulation is more specific and more closely dard technique (30,31), and operations were performed under simulates the physiological conditions of the aforementioned aseptic conditions. In brief, mice were euthanized under CD147‑related T cell stimulation signaling pathway, which general anesthesia. Full‑thickness skin grafts 1 cm in diameter could explain the difference between two groups. To further were obtained from the tails of C57BL/6 (H‑2b) donor mice, evaluate whether the inhibitory effects were mediated by the transplanted onto the backs of BALB/c (H‑2d) recipient mice, cytotoxicity of 5A12, peripheral CD4+ T cells were stained and protected by a bandage for 9 days. The transplanted grafts with Annexin V and PI. Even at the highest concentration were scored visually daily, and a graft was considered fully (1 mg/ml), the addition of 5A12 did not significantly affect rejected when 80% of the tissue was necrotic. Drugs were the rate of apoptosis (Annexin V+/PI‑ and Annexin V+/PI+) administered to the mice every day via intraperitoneal injec- (Fig. 1C), indicating that the effects of 5A12 were not mediated tion (antibodies, 5 mg/kg; FK506, 0.5 mg/kg) from the day by apoptosis. of the grafting (D0) to the day of rejection. The mice in the autograft and allograft control groups were injected with an 5A12 inhibited the one‑way MLR. The one‑way MLR equal volume of saline. Mice from each group were randomly system is an effective method for evaluating an immunosup- selected and euthanized under general anesthesia on D10. pressor in vitro. In the present study, we used it to analyze 4596 LUAN et al: CD147 BLOCKADE SHOWS GREAT POTENTIAL FOR THE TREATMENT OF GRAFT REJECTION

Figure 1. 5A12 inhibited the proliferation of CD4+ T cells isolated from human peripheral blood without causing toxicity. Cells were stimulated with PHA (A) or CD3/CD28 magnetic beads (B) in the presence of 5A12 for 96 h. The addition of 5A12 to the cultures strongly inhibited proliferation (A and B). CD4+ T cells were stimulated with PHA for 96 h in the presence of 5A12 at 1, 0.5, 0.1, 0.05, 0.01, or 0.001 mg/ml, and the level of apoptosis was evaluated by flow cytometry after staining with FITC‑conjugated Annexin V and PI (BioLegend) (C). Data are representative of three independent experiments. Multiple comparisons were performed with one‑way ANOVA ***P<0.001.

the immunosuppressive activity of 5A12; the protocol is included in the Materials and Methods section. When two MHC‑mismatched lymphocytes are mixed, they will each promote the proliferation of the other. Lymphocytes incubated with mitomycin C lost their multiplication capacity and were used as stimulator cells. As shown in Fig. 2, 5A12 significantly inhibited the proliferation of the responder cells, i.e., the PBMCs, nearly to the point of equivalency with the baseline, unstimulated level. The minimum significant concentration was 200 µg/ml (data not shown). As the balance between Th cell subsets plays an important role in the development of graft rejection, we assessed the influence of 5A12 on expression of the Th1/Th2/Th17 cell‑related cytokines IFN‑γ, IL‑4 and IL‑17 from the culture supernatants. However, the addition Figure 2. 5A12 inhibited the one‑way MLR. PBMCs isolated from human of 5A12 had no influence on the expression of these cytokines. volunteers served as responder cells, and splenocytes isolated from C57BL/6 mice served as stimulator cells. Cells were suspended in RPMI 1640 supplemented with 10% fetal bovine serum. The suspended cells were Establishment of the allogeneic skin transplantation mouse seeded at a density of 1.5x105 cells/well in round‑bottomed 96‑well plates model. To further clarify the immunosuppressive effects and cultured with or without 5A12 for 96 h; then, lymphocyte proliferation and underlying mechanisms, we assessed the in vivo effects was measured using a CCK‑8 assay. Data are representative of three inde- of CD147 blockade using an allogeneic skin transplantation pendent experiments. Multiple comparisons were performed with one‑way A NOVA *P<0.05, **P<0.01. P: PBMCs, C: C57BL/6 mouse splenocytes, MC: mouse model. Full‑thickness skin grafts were obtained from b Mitomycin‑treated C57BL/6 mouse splenocytes. the tails of C57BL/6 (H‑2 ) donor mice and transplanted onto MOLECULAR MEDICINE REPORTS 16: 4593-4602, 2017 4597

Figure 3. Establishment of the allogeneic skin transplantation model. Full‑thickness skin grafts 1 cm in diameter were obtained from the tails of C57BL/6 (H‑2b) donor mice and transplanted onto the backs of BALB/c (H‑2d) recipient mice. On the tenth day after transplantation, the bandages were removed, and the skin grafts were observed and imaged every day thereafter. The images shown above were randomly chosen from the autologous transplantation (A) and allogeneic skin transplantation groups (B).

new hair, indicating that the transplantation operation was successful (Fig. 3A). In contrast, grafts in the allogeneic skin transplantation group began to show signs of inflammation on the 10th day after transplantation. Due to rejection, the grafts turned red, and a yellow exudate was present around them. Over time, the skin tissue began to lose activity and become stiffer. By the 12th and 13th days, the skin grafts were either fully necrotic or had been rejected. Subsequently, scar tissue began to replace the wound (Fig. 3B).

CD147 blockade prolonged the survival of transplanted Figure 4. Blockade of CD147 with RL73 prolonged skin allograft survival. skin. We next assessed the in vivo effects of CD147 blockade Skin allografts were transplanted from C57BL/6 mice to BALB/c recipients, using the allogeneic skin transplantation mouse model and a which were then intraperitoneally injected with RL73, its isotype control commercially functional anti‑mouse CD147 antibody (RL73). (n=6), FK506 or equal volumes of saline (n=8) every day from day 1 until complete rejection. Isogeneic skin transplantation was used as a control To better evaluate the immunosuppressive effect of the anti- (n=8). Graft survivals were analyzed by the Kaplan‑Meier method using the body, we included a control group treated with a common log‑rank test. RL73 and FK506 prolonged graft survival compared with that effective immunosuppressant, Tacrolimus (FK506), which of the isotype control and saline groups (14 vs. 13 days; P<0.0001). strongly inhibits T cell proliferation. From the day of the skin graft operation, these drugs were administered to the mice every day via intraperitoneal injection (RL73 and its isotype the backs of BALB/c (H‑2d) recipient mice. On the tenth day control, 5 mg/kg; FK506, 0.5 mg/kg). The mouse weight after transplantation, the bandages were removed, and the skin of each group did not differ, except for the FK506‑treated grafts were observed every day. In the autologous transplanta- mice, which showed slight weight loss compared with the tion group, the status of the skin grafts improved over time. other groups (data not shown). As shown in Fig. 4, mice that On the 15th day after transplantation, the grafts began to grow received RL73 and FK506 had a mean graft survival time of 4598 LUAN et al: CD147 BLOCKADE SHOWS GREAT POTENTIAL FOR THE TREATMENT OF GRAFT REJECTION

Figure 5. CD147 blockade decreased the level of inflammatory cell infiltration in transplanted skin. Skin allografts were transplanted from C57BL/6 mice to BALB/c recipients, which were then intraperitoneally injected with RL73, its isotype control (n=6), FK506 or equal volumes of saline (n=8) every day. Isogeneic skin transplantation was used as a control (n=8). Mice from each group were randomly selected and euthanized under general anesthesia on D10. Skin grafts were harvested and fixed in 4% formalin. Histological analysis of the skin grafts was performed by staining 5‑mm sections of paraffin‑embedded tissues with hematoxylin and eosin. Representative histological results are shown (A), magnification, x200. Inflammatory cells were counted by examining 10 different visual fields under a microscope at 400x magnification in a blinded manner (B). Multiple comparisons were performed with one‑way ANOVA ***P<0.001.

14 days (range: 13‑15 days, P<0.05) compared with saline‑ transplantation. The serum expression levels of IL‑4 and and isotype control antibody‑treated mice (range: 12‑14 days). IFN‑γ were not significantly different among the groups, Although the mean survival time was prolonged by only one although the serum level of IL‑4 was slightly increased in the day, RL73 showed the same behavior as FK506 in skin trans- RL72‑ and FK506‑injected mice. However, the serum level of plantation. IL‑17 was significantly decreased by RL73 to below the levels observed in normal mice (Fig. 6). CD147 blockade decreased the level of inflammatory cell infiltration in transplanted skin grafts. We then assessed the CD147 blockade decreased the proportions of CD4+ and level of inflammatory infiltration in the skin grafts on the tenth CD8+ memory T cells in recipient mouse peripheral blood. day after transplantation; the rejection response is believed To further elucidate the immunosuppressive effects of the to be the strongest during this time. Hematoxylin‑eosin CD147 antibodies and examine the altered IL‑17 levels, on the staining showed there was little inflammatory infiltration in 10th day after transplantation, the proportions of T cell subsets the normal skin and grafts of RL73‑treated mice. Next, we within the peripheral blood were detected in the skin graft counted the infiltrated inflammatory cells and found that in the recipients who received different treatments. We found that RL73‑treated group, the number was 75% lower than that in the the proportions of CD4+ and CD8+ T cells were decreased by isotype control‑treated group. Similarly, the number of infil- FK506 but not by RL73. Then, we assessed the proportions of trated inflammatory cells was 18% lower in the FK506‑treated Th1/Th2 and Th17/Treg cells. In the FK506‑ and RL73‑treated mice than in the saline‑treated mice, but this result was not groups, the levels of Th2 cells were slightly increased, but this significantly different (P=0.07) (Fig. 5A and B). result was not significant. As memory T cells are confirmed to be involved in development of the rejection reaction and to CD147 blockade inhibited IL‑17 expression in the serum of play an important role, we detected the proportions of both skin graft recipients. We also tested the serum levels of IFN‑γ, CD4+ and CD8+ memory T cells and found that they were IL‑4 and IL‑17 in skin graft recipients on the 10th day after sharply decreased (Fig. 7A and B). Compared with the isotype MOLECULAR MEDICINE REPORTS 16: 4593-4602, 2017 4599

Figure 6. CD147 blockade inhibited serum IL‑17 expression in skin graft recipients. Skin allografts were transplanted from C57BL/6 mice to BALB/c reci pients, which were then intraperitoneally injected with RL73, the RL73 isotype control (n=6), FK506 or equal volumes of saline (n=8) every day. Mice from each group were randomly selected and euthanized under general anesthesia on D10 post‑treatment. Blood was then collected from the orbital sinus. Plasma was obtained by centrifugation, and the serum levels of the cytokines IFN‑γ (A), IL‑4 (B) and IL‑17 (C) were measured using a MILLIPLEX MAP kit. Multiple comparisons were performed with one‑way ANOVA **P<0.01. control, the proportion of CD4+ memory T cells decreased resistant to immunosuppressive therapies. In our preliminary to 70%, and that of CD8+ memory T cells decreased to 80% experiments, we used FK506, a clinically effective immuno- (Fig. 7C and D). We also tested the proportions of the above suppressant, to score graft survival time and found that it was T cell subsets within the spleen and found that the decreases not particularly effective at the common doses used in clinical in their proportions were not as significant as the decreases in treatment, from 0.01 to 0.3 mg/kg/day. Then, higher doses of their proportions in the peripheral blood; however, the propor- FK506, from 0.01‑0.65 mg/kg/day, were tested. FK506 was tion of CD4+ memory T cells in the spleen was still decreased ineffective at doses less than 0.5 mg/kg/day. Administering compared with that in the peripheral blood. FK506 at a dose of 0.5 mg/kg/day prolonged the mean graft survival time by one additional day compared with doses Discussion less than 0.5 mg/kg/day (P<0.001); however, the mice began to show slight weight loss at this dose. At doses of more The specific inhibition of CD147 via blocking antibodies has than 0.5 mg/kg/day, more obvious, serious side effects were been successfully tested in models of T cell‑related diseases, observed. To elevate the efficacy of anti‑CD147, we repeated including autoimmune diseases (e.g., RA (31‑34) and multiple the experiment in the same system. sclerosis (MS) (35,36) and GVHD following bone marrow In our study, the anti‑mouse CD147 antibody exhibited transplantation (19,25,27,37). However, there have been no the same effect as FK506 on prolonging skin graft survival reports on this topic in the context of organ transplantation. and caused fewer side effects. In addition, the antibody Skin allograft rejection model is often characterized by mitigated inflammatory cell infiltration in transplanted skin acute rejection mediated by cellular immunity. It is widely and decreased the serum level of IL‑17, a factor that plays used in basic pharmacodynamic studies or new methods for an important role in initiation of the inflammatory response. treating graft rejection reaction (38‑40). Compared with other IL‑17 can promote the release of pro‑inflammatory cytokines, allograft models, such as heart or islet transplantation, the skin thereby amplifying the inflammatory response (41). We found allograft rejection model has the advantage of being simple that the blockade of CD147 had no obvious influence on the to create and easy to observe. On the other hand, the skin proportions of Th17/Treg cells in the peripheral blood of allograft rejection model is an aggressive model that is often recipient mice, although the proportion of Th2 cells showed 4600 LUAN et al: CD147 BLOCKADE SHOWS GREAT POTENTIAL FOR THE TREATMENT OF GRAFT REJECTION

Figure 7. CD147 blockade decreased the proportions of CD4+ and CD8+ memory T cells within recipient peripheral blood. Skin allografts were transplanted from C57BL/6 mice to BALB/c recipients, which were then intraperitoneally injected with RL73, the RL73 isotype control (n=6), FK506 or equal volumes of saline (n=8) every day. Mice from each group were randomly selected and euthanized under general anesthesia on D10 post‑treatment. Peripheral blood lymphocytes were stained with antibodies, according to conventional treatment methods, and then immediately detected using an LSRFortessa X‑20 flow cytometer (BD Biosciences, USA). Representative flow cytometry results for the CD4+ memory T cells (CD4+ CD44 high CD62 L low) and CD8+ memory T cells (CD8+ CD44 high CD62 L low) are shown (A and B). The mean values of the CD4+ memory T cell (C) and CD8+ memory T cell proportions were calculated (D). Nonparametric Mann‑Whitney U tests were performed *P<0.05, ***P<0.001. 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